See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/232695664 Calcium distribution and function in the glandular trichomes of Lavandula pinnata L Article in Journal of the Torrey Botanical Society · April 2010 DOI: 10.3159/09-RA-046.1 CITATIONS READS 5 103 2 authors, including: Jing-Ping Liao Chinese Academy of Sciences 45 PUBLICATIONS 336 CITATIONS SEE PROFILE Some of the authors of this publication are also working on these related projects: Living Collections of Botanical Gardens View project All content following this page was uploaded by Jing-Ping Liao on 13 November 2016. The user has requested enhancement of the downloaded file. Journal of the Torrey Botanical Society 137(1), 2010, pp. 1–15 Calcium distribution and function in the glandular trichomes of Lavandula pinnata L. Shan-Shan Huang1,2 The Institute of Chinese Medicine of Guangdong Province, Guangzhou, 510520, China Jing-Ping Liao South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, 510650, China Bruce K. Kirchoff University of North Carolina at Greensboro, Department of Biology, Greensboro, NC 27402 HUANG, S-S. (The Institute of Chinese Medicine of Guangdong province, Guangzhou, 510520, China), J.-P. LIAO (South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, 510650, China). AND B. K. KIRCHOFF (University of North Carolina at Greensboro, Department of Biology, Greensboro, NC 27402), Calcium distribution and function in the glandular trichomes of Lavandula pinnata L. J. Torrey Bot. Soc. 137: 1–15. 2010.—Calcium distribution during peltate and capitate glandular trichome development in Lavandula pinnata L. was examined with the potassium antimonate precipitation method. In order to establish a role for calcium in the secretory process and elucidate calcium function in the glands, the effects of calcium removal were investigated by treatment with nifedipine (Nif, a calcium channel blocker) and Ethylene glycol-bis (2-aminoethyl ether)-N, N, N9,N9-tetraacetic acid (EGTA, a calcium chelator). Untreated, mature glands accumulate many calcium precipitates in the subcuticular space and adjacent cell wall during secretion. In Nif or EGTA treated plants these precipitates disappear, and the amount of secretory product is drastically reduced. Calcium removal also results in a reduction in gland density, cells with decreased cytoplasmic density, formation of a lax cell wall, abnormal formation of the subcuticular space, thinning of the cuticle, and the presence of multivesicular bodies near the plasma membrane. At the post-secretory stage, calcium precipitates are common on the degenerating organelles. These results support a role for calcium in gland development, secretion, and programmed cell death. Key words: antimonate precipitation, calcium, EGTA, glandular trichome, Lavandula pinnata L., nifedipine, secretion, ultrastructure. Calcium is an essential element in biological 1996, Taylor and Hepler 1997, Franklin-Tong systems. In its ionic form (Ca2+) it has been 1999, Eckardt 2001, Plieth 2001, Campanoni shown to be crucial in the control of numerous and Blatt 2007). Removal of calcium from the physiological and developmental processes in nutrient supply results in rapid death of cells in plants (Bush 1995, Zhang and Cass 1997, the apical meristem, and a cessation of growth Trewavas and Malho´ 1998, Sanders et al. (Epstein 1972, Gilroy et al. 1993). 1999, Scrase-Field and Knight 2003, Medve- Pollen tubes have been found to respond to dev 2005, Wheeler and Brownlee 2008). For calcium gradients by elongating toward higher instance, the Ca2+ ion is involved in intracel- Ca2+ concentrations in in-vitro assays (Mas- lular events such as protoplasmic streaming, carenhas and Leonarad 1962, Pierson et al. nuclear division, cell division, cell plate for- 1994). When calcium channel blockers are mation, and polarized growth in rhizoids and added to the medium, pollen tube growth is pollen tubes (Takagi and Nagai 1983, Sakai- restrained, suggesting that calcium is acting as Wada and Yagi 1993, Malho´ and Trewavas an intercellular signal (Picton and Steer 1985, Pierson et al. 1994, Herrmann and Felle 1995, Fan et al. 1996, Carol et al. 1997). Consistent 1 We thank the National Natural Science Foun- with this view, elevated cytosolic Ca2+ concen- dation of China (39870087, 30370099, 40332021), trations ([Ca2+] ) have been reported to drive and the Key Scientific Item of Guangdong c (045C013) for financial support. Xing-Lan Xu and docking and fusion of exocytotic vesicles at the Xiao-Ying Hu, instructors in the Electron Micro- tip of pollen tubes (Carol et al. 1997). In a scopy facility of the South China Botanical Garden, related study, Roy et al. (1999) found positive assisted the first author in learning EM. feedback between Ca2+ concentration and 2 Author for correspondence. E-mail: hss@scib. ac.cn exocytosis in lily pollen tubes. According to Received for publication August 6, 2009, and in this work, high concentrations of calcium revised form January 7, 2010. promote exocytosis, thus providing more cell 1 2 JOURNAL OF THE TORREY BOTANICAL SOCIETY [VOL.137 wall substance and causing the circumference As in other Lamiaceae, Lavandula pinnata of the cell membrane to increase, which in turn possesses two kinds of glandular trichomes causes more calcium channels to open (Roy et (Ascensa˜o and Pais 1998, Gersbach 2002, al. 1999). Huang et al. 2008). Both capitate and peltate Calcium levels in the anther have also been trichomes are initiated from a single proto- studied and have been found to change with dermal cell, and undergo several cell divisions the stage of development. For instance, when to produce stalk and head cells. However, visualized with potassium antimonate precipi- while there are one to two secretory cells in the tation in photoperiod-sensitive male sterile head of a capitate trichome, there are eight rice, calcium precipitates accumulate in the cells in the head of a peltate trichome (Huang anther walls, on the surface of pollen grains, et al. 2005, 2008). In both cases, gland and in Ubish bodies, but they are less frequent development is very rapid (approximately on the pollen surface at the late microspore 60 h) in comparison to leaf expansion (18– stage (Tian et al. 1998). Later, as starch 25 d, Huang et al. 2008). Three different accumulates in the mature pollen grains, the secretory stages can be recognized: presecre- number of calcium precipitates decreases in tory, secretory and post-secretory (Ascensa˜o these cells and increases in the parenchyma- and Pais 1998, Turner et al. 2000, Huang et al. tous cells of the connective tissue (Tian et al. 2005, 2008). The mechanism of secretion is 1998). Changes in calcium distribution during similar to that of other Lamiaceae (Huang et development have also been reported in al. 2008). developing ovules of Nicotiana tabacum (Tian In this study, potassium antimonite precipi- and Russell 1997), pollen grains of Chlorophy- tation is used to investigate loosely bound tum elatum (GO´ rska-Brylass et al. 1997), calcium distribution during both capitate and Torenia fournieri (Chen et al. 2008), and peltate glandular trichome development. The during pollen tube growth of tobacco (Xie et calcium channel blocker nifedipine (Nif) and al. 2005a). the calcium chelator Ethylene glycol-bis (2- Calcium has also been implicated in the aminoethyl ether)-N, N, N9,N9-tetraacetic function of the stylar transmitting tract (Zhao acid (EGTA) are used to elucidate calcium et al. 2004), dividing root cells (Vaughan et al. function during gland development (Williams 1987, Sakai-Wada and Yagi 1993) and poten- 1970, Reiss and Herth 1985, Rosales and tially as a causative factor in Blossom-end rot Brown 1992, Fan et al. 1996, Moysset and of tomato (Suzuki et al. 2003). Despite what is Simo´n 1990, Zhang et al. 2000). Using these known from these studies, a role for calcium in calcium antagonists, we investigate the effects the secretion of essential oils has not been of calcium on trichome number, morphology, established. development, and on the secretory process The glandular trichomes of the Lamiaceae itself. are a well-established source of essential oils. Two types of trichomes occur in the family. Material and Methods. MICROPROPAGATION These trichomes are termed peltate or capitate OF APICAL SHOOTS.PlantsofLavandula pinnata depending on the structure of the secretory L. were grown in a greenhouse at the South head (Werker 1993). In both cases, the China Botanical Garden, Guangzhou, China. secretory product accumulates in a subcuticu- Apical shoot explants for the study of calcium lar space outside of the cell wall. The peltate distribution were prepared according to the trichomes produce most of the essential oil, procedure of Huang et al. (2008). Trichome with terpenes comprising the main component development was studied under four growth (Clark et al. 1997, Turner et al. 2000). The conditions: light-grown control, dark-grown capitate trichomes secrete varying amounts of control, 100 mM Nif (maintained in the dark), polysaccharides in addition to some terpenes and 5 mM EGTA (maintained in the light). (Werker 1993). Prior to secretion, the tri- The dark-grown control was necessary be- chomes undergo several cell divisions, and the cause Nif is inactivated by light, so Nif treated cuticle thickens. The subcuticular space forms plants must be maintained in the dark between the cuticle and the cell wall, and (Dentinger 2003). enlarges as the essential oil is secreted (Ascen- sa˜o et al. 1997, Turner et al. 2000, Gersbach CALCIUM VISUALIZATION. Calcium is present 2002, Huang et al. 2008). in plant tissues in free (ionic), loosely bound, 2010] HUANG ET AL.: CALCIUM IN THE TRICHOMES OF LAVANDULA PINNATA L. 3 and bound states. Visualization of each pool is caused the leaf margins to dry, and some buds strongly technique dependent (Ge et al. 2007). to stop growing. When [Nif] was increased to Although a precise microspectrofluorometric 250 mM, the leaves began to dry out after method is available to measure free cytosolic 10 days and the seedlings died after 15 days.